Rapid Consumption of Dihydrogen Injected into a Shallow Aquifer by Ecophysiologically Different Microbes

The envisaged future dihydrogen (H2) economy requires a H2 gas grid as well as large deep underground stores. However, the consequences of an unintended spread of H2 through leaky pipes, wells, or subterranean gas migrations on groundwater resources and their ecosystems are poorly understood. Therefore, we emulated a short-term leakage incident by injecting gaseous H2 into a shallow aquifer at the TestUM test site and monitored the subsequent biogeochemical processes in the groundwater system. At elevated H2 concentrations, an increase in acetate concentrations and a decrease in microbial α-diversity with a concomitant change in microbial β-diversity were observed. Additionally, microbial H2 oxidation was indicated by temporally higher abundances of taxa known for aerobic or anaerobic H2 oxidation. After H2 concentrations diminished below the detection limit, α- and β-diversity approached baseline values. In summary, the emulated H2 leakage resulted in a temporally limited change of the groundwater microbiome and associated geochemical conditions due to the intermediate growth of H2 consumers. The results confirm the general assumption that H2, being an excellent energy and electron source for many microorganisms, is quickly microbiologically consumed in the environment after a leakage.


■ INTRODUCTION
The dihydrogen (H 2 ) economy is an aspired future in which H 2 is used as an energy carrier and raw material and therefore requires both a gas network for transport and storage options. 1,2−7 The risks of geological H 2 storage technologies have not yet been fully investigated although leakages of stored gas through geological fractures, for instance, have been reported regularly at underground gas storage (UGS) sites where gas mixtures containing up to 95% H 2 can be stored. 8,9iogenic H 2 is mainly produced by dark fermentation of organic substrates in anoxic soil habitats; however, concentrations are usually low due to the immediate consumption of generated H 2 by anaerobic hydrogenotrophs already in the anoxic areas or aerobic hydrogenotrophs at oxic−anoxic interfaces. 8,10,11Both bacteria and archaea belong to these hydrogenotrophs, which use H 2 as an electron donor to generate energy.Generally, hydrogenase, a metal-containing enzyme that exists in different forms, catalyzes the conversion of H 2 into protons and electrons (as well as the reverse reaction) in all hydrogenotrophic microorganisms. 12Relevant electron acceptors of hydrogenotrophs with decreasing energy yield are oxygen (O 2 ) as well as nitrate (NO 3 − ), nitrite (NO 2 − ), ferric iron (Fe 3+ ), sulfate (SO 4 2− ), and carbon dioxide/bicarbonate (CO 2 /HCO 3 − ). 13 The latter refers to hydrogenotrophic methanogenesis and acetogenesis via H 2 oxidation and CO 2 /HCO 3 − reduction.During anaerobic digestion of organic matter (OM), H 2 is an important electron carrier of reducing equivalents which are transmitted directly between cells of H 2 producers and consumers (termed interspecies H 2 transfer), facilitating syntrophic interactions. 10,14Since hydrogenotrophy is widely distributed within different taxonomic and ecophysiological groups, hydrogenotrophic prokaryotes can be common in aquifers.Many hydrogenotrophs are able to switch their energy metabolism and grow on other (organic) substrates as well to ensure survival when H 2 is not available. 10,11he most relevant microbial H 2 -consuming processes expected at H 2 -UGS sites are methanogenesis, sulfate reduction, as well as acetogenesis, which can possibly occur simultaneously (in different microenvironments) when H 2 is stored in high concentrations. 9,15,16Gas migrations from deep reservoirs or leakages in gas pipelines into the shallow subsurface could potentially affect shallow groundwater resources by H 2 -oxidation processes and stimulate microbial activity.The production of toxic and corrosive dihydrogen sulfide (H 2 S) by H 2 -driven microbial sulfate reduction is of particular importance. 8Furthermore, the production of methane (CH 4 ) may partly foil the climate protective usage of H 2 because CH 4 is a strong greenhouse gas.Previous laboratory studies examining the biogeochemical effects of millimolar H 2 concentrations 15,17,18 found Fe 3+ and sulfate reduction as well as acetogenesis as predominant H 2consuming processes under anoxic conditions.In a previous in situ study in the Opalinus Clay formation at the rock laboratory Monte Terri, the response of a deep subsurface microbial community to gaseous H 2 was investigated, demonstrating H 2 oxidation by different ecophysiological groups. 19However, environmental conditions of the Opalinus Clay differ from those normally found in shallower areas, e.g., due to different temperatures, geologic textures, pressures, and salinities. 9Thus, it is unknown whether the results of this study are transferable to shallower aquifers.Recently, a short H 2 leak was simulated in a shallow unconfined aerobic chalk aquifer in France by injecting H 2 -saturated groundwater; notably, microbial consumption of H 2 was not observed in this field study due to the limited amount of dissolved H 2 added (9 g in total), a rapid transfer of the dissolved H 2 plume through the aquifer, and its significant dilution downstream of the injection well. 20he aim of this field-scale study was to assess the effects of a gaseous H 2 leakage from, e.g., a UGS on the groundwater microbiome in a shallow aquifer by a controlled three-day H 2release experiment.Stable isotope effects associated with H 2 injection, H 2 transport processes, and putative microbial consumption in the course of the field experiment have recently been described. 18Here, we determined the time response and mode of microbial H 2 consumption until recovery from initial biogeochemical conditions.To the best of our knowledge, the effects of the entry of gaseous H 2 on a groundwater microbiome and the associated redox reactions in a shallow aquifer have not been investigated in situ so far.

■ MATERIALS AND METHODS
Experimental Site and Setup.The hydrogeological TestUM test site is located near Wittstock/Dosse (Brandenburg, Germany) approximately 100 km from Berlin (Figure S1).Previously, a CO 2 injection test 21−24 and a five-day infiltration test of ∼75 °C hot water 25−27 were conducted in other areas of the test site (Figure S1).The corresponding studies include a detailed description of the location and give an overview of the wider geological conditions.The geological conditions in the area of the gaseous H 2 injection test as well as the injection methodology are summarized in the Supporting Information (SI) and were also described in our previous study in which we reported on the stable isotope effects associated with the H 2 injection. 18roundwater Sampling.Groundwater was sampled as previously described; 18,27 a detailed description is given in the Supporting Information (SI-1−3).The sampling wells were selected according to a previous study for monitoring isotopic effects, 18 i.e., groundwater was taken from 11.5, 14.5, or 17.5 m from wells D04, D06, and D11 and subsequently analyzed, respectively.Wells D04 and D06 are located in close vicinity (<5 m) and downstream of the injection spots, whereas well D11 is located around 25 m southeast of the injection, functioning as a H 2 -free control (Figure S1B).The initial states of the hydrogeochemical conditions and the groundwater microbiome were determined 19−20 days before the H 2 injection (henceforth referred to as T0 sampling).Groundwater samples were taken on days 2 and 3 of the three-day H 2 injection.Post injection (henceforth referred to as postinjection phase), groundwater was sampled after 1 day (solely from well D06), and after 5−6, 13−14, 19−20, and 26−27 days.When the H 2 concentrations fell below the detection limit (henceforth referred to as postincident phase), further groundwater samples were collected, specifically, 76−77, 214, and 243−244 days after the injection.
Each sample used for 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing consisted of 4 L of groundwater.For total cell counting, 5 mL of groundwater was fixed on the site with 5 mL (vol/vol) of 4% paraformaldehyde (PFA) in a sterilized 50 mL serum bottle that was sealed gastight with sterilized butyl rubber stoppers and aluminum crimp seals as described in detail elsewhere. 26All samples for microbiological analyses were taken from 14.5 m well depth and kept at around 4 °C in a fridge on-site (maximum 2 days) and transported cooled to the Helmholtz Centre for Environmental Research in Leipzig (Germany), where they were processed within 3 days.
To gain the amount of potential electron acceptors in the solid phase, the reactive Fe 3+ content of 15 sediment samples from the injection horizon of wells U03 and U05 was determined by extraction with 1 M HCl as described in Leventhal and Taylor 28 and subsequent photometric analyses of the extracts using the ferrozine method, 29 whereby hydroxylammonium chloride functions as a reductive agent.
Amplicon Sequencing.The samples for the microbial diversity analyses were processed as previously described. 26he V3−V4 hypervariable region of the 16S rRNA gene was amplified according to Klindworth et al. 30 using a 2× MyTaq Mix (Bioline; Heidelberg; Germany), whereby the primer pair S-D-Bact-0341-b-S-17−S-D-Bact-0785-a-A-21 was chosen due to its good bacterial diversity coverage. 30The libraries for sequencing were prepared as described in the Illumina 16S Metagenomic Sequencing Library Preparation protocol. 31The pool with 4 nM libraries was run on an Illumina MiSeq system (Illumina; CA) using v3 600 cycles chemistry.
The paired-end, demultiplexed fastq files were analyzed utilizing Quantitative Insights Into Microbial Ecology 2

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(QIIME 2) version 2021.11 32and a pipeline that was initially provided by Dr. Denny Popp (University of Leipzig Medical Center; Germany) but modified to analyze this data set.The primer sequences were removed, and untrimmed reads as well as reads <50 base pairs (bp) were discarded using cutadapt version 2.10. 33Denoising was done with the QIIME 2 DADA2 plugin; this included trimming (at positions 284 and 202 of the forward and reverse reads, respectively), quality filtering, learning of the error rates, dereplicating, chimera removal, and merging of paired-end reads. 34Since the samples were sequenced on two Illumina MiSeq runs, DADA2 was run separately on data obtained from the respective run.The denoised data were merged before taxonomic annotation using the SILVA 138 (99%) data set, 35,36 which had been trained with the primer pair used to amplify the V3−V4 hypervariable region of the 16S rRNA gene in advance.Reads classified as chloroplasts as well as mitochondria were removed (reads classified as Eukaryota or unassigned reads were not present in the data set).16S rRNA gene sequences were deposited at the European Nucleotide Archive (ENA) under accession number PRJEB56759.
Total Cell Counting.The methodology was used for total cell counting has been described in Keller et al. 26 Cells were counted manually using ImageJ 1.53e along with the cell counter plugin.
Figures and Statistical Analyses.Figures and statistical analyses were conducted with RStudio version 2022.02.1.+461. 37The detailed procedure including all used functions and packages is described in the SI (SI-1).The Shannon− Wiener indices were used as a proxy for the α-diversity, and to illustrate the β-diversity, nonmetric multidimensional scaling (NMDS) was performed.

■ RESULTS AND DISCUSSION
Hydrogeochemical Changes Due to H 2 Injection.Data of geochemical and physicochemical parameters in groundwater samples from wells D04, D06, and D11 at different depths (11.5, 14.5, and 17.5 m) before and after H 2 injection are shown in Figures S2−S6.Injection of gaseous H 2 into the saturated zone of the aquifer is expected to result in the fast dispersion of gas-phase H 2 through channels in different directions, accompanied by stripping of other gases, displacement of water, and slow dissolution of H 2 in the aqueous phase. 18During H 2 injection, the aqueous H 2 concentrations increased to up to 600 μM in well D04 at 14.5 m depth, whereby similar amounts were detected at 17.5 m depth (Figure S2A).The aqueous H 2 concentrations of well D06 generally rose until 19−20 days post injection when the highest values of around 830 μM were observed at 11.5 and 14.5 m depths (Figure S2E).76−77 days post injection and afterward (until the last sampling for this study), H 2 was no longer detectable in any groundwater samples at any depths of wells D04 and D06 (Figure S2A,B), respectively.No H 2 was detected in the control well D11 throughout the whole monitoring period (Figure S2I).
H 2 injection went along with low oxygen concentrations (Figure S6C,F) and sharply decreasing redox potentials to −121 mV in well D04 (Figure S2B) and to −249 mV in well D06 (Figure S2F), indicating the rapid development of more reducing redox conditions in both wells.In well D04, the redox potential at 14.5 m depth stayed at negative values in the course of the experiment, whereas in well D06, the redox potential increased to +164 mV 43 days after H 2 injection and slowly decreased to negative values afterward.Initial reducing redox conditions in wells D04 and D06 upon H 2 injection were also indicated by increased concentrations of dissolved iron species (Figure S6A,D).In contrast, redox potentials of the control well D11 were always positive, and dissolved iron species were below the detection limit; however, redox potentials sharply decreased 20 days after H 2 injection from around +400 mV to less than +100 mV within a week, but increased later on to values of above +300 mV, which can probably be attributed to a combination of groundwater displacement and mixing processes induced by gaseous H 2 injection.The control well D11 might be influenced as well by groundwater being displaced from the injection area by preferential flow in the southeast direction; 18 this assumption is supported by the appearance of small amounts (<15 nM) of trichloroethene (TCE) at all depths of the control well D11 in the early postinjection phase and later on (Figure S4L), indicating mobilization of TCE-containing groundwater from other aquifer areas.TCE was detected already before H 2 injection at some depths of wells D04 and D06 (Figure S4D,H).Chlorinated aliphatics were probably introduced into the aquifer during the usage of the site as a military airport in the former Germany Democratic Republic. 21,22he concentrations of major cations (Na + , Ca 2+ , Mg 2+ ) and anions (SO 4 2− , Cl − ) were predominantly stable during and after H 2 injection (Figures S3 and S5).Notably, NO 3 − was not detectable at all in well D04 and below 40 μM in two depths of well D06 (11.5, 14.5 m) before H 2 injection; NO 3 − quickly disappeared after H 2 injection in well D06 and returned after 78 days at 11.5 m depth at similar concentrations as detected before H 2 injection, but it was detected only sporadically at 14.5 and 17.5 m depths in the postinjection phase (Figure S3E).− during and after H 2 injection (Figure S3I), indicating mixing processes.At a depth of 17.5 m, some NO 2 − (<30 μM) was detected before and after H 2 injection (Figure S3J), indicating ongoing microbial NO 3 − reduction.In our previous study, we observed an equilibrium isotope fractionation of H 2 in the course of its depletion, which was interpreted to be caused by microbial oxidation due to the enzyme hydrogenase; 18 this hypothesis is supported by the transient increasing concentrations of acetate and formate in wells D04 and D06 in the postinjection phase (Figure S4A,B,E,F) and the absence of these compounds in the control well D11 (Figure S4I).Formate is a metabolite and acetate is an end product of the microbial conversion of H 2 and CO 2 upon homoacetogenesis; 38 acetate formation from H 2 and CO 2 is usually accompanied by a transient accumulation of formate. 39CH 4 , indicative of methanogenesis, was detected above background concentrations in the early postinjection phase once in well D04 (Figure S4C), but at the same time also in the control well D11 (Figure S4K).Hence, geochemical data did not indicate a relevant H 2 -driven methanogenesis in this phase.Higher CH 4 concentrations (up to 27 μM) were sporadically observed in the late postinjection phase in deeper zones of well D06 (Figure S4G) and were not linked to more negative redox conditions (Figure S2F) typical for methanogenesis; we cannot rule out that CH 4 was formed in other areas of the site and transported to well D06.

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In summary, the hydrogeochemical and physicochemical data show that the aquifer was saturated with H 2 close to the injection well, accompanied by slightly changing hydrogeochemical conditions.A part of these data and previously reported stable isotope data indicated that H 2 was microbiologically consumed in the aquifer and that at least a part of the H 2 was oxidized by homoacetogens.To obtain a more detailed view of the microbiological processes connected to the injection of H 2 , we frequently determined the composition of the microbial communities of the H 2 -exposed wells D04 and D06 and the control well D11 before, during, and after H 2 injection.

Microbial Diversity Changes Due to H 2 Injection.
Generally, due to the multidirectional spatial sampling (especially in 2 in.wells) and the volume of 4 L, each sample could capture a mixture of different microenvironments in the groundwater and might not represent the microbial community in a specific location with constant conditions.Since a highresolution sampling of individual niches was not feasible, the samples represent a broader picture of the alterations in the groundwater microbiome.
Changes of α-and β-Diversity in D04, D06, and D11.The Shannon−Wiener indices, representing the α-diversity in the investigated groundwater microbial communities, were be-  Environmental Science & Technology tween 4.4 and 5.8 (5.4 ± 0.6) in the T0 samples (Figure 1).In well D04, the mean values were the same before (4.5 ± 0.1) and during (4.5 ± 0.2) the H 2 injection, decreased to 3.4 ± 0.4 post injection, and increased (3.9 ± 1.3) again in the postincident phase, whereby, in this phase, they reached both their minimum (2.4) and their maximum (4.8) 76−77 and 214 days after the H 2 injection, respectively.The highest Shannon−Wiener index of 5.8 was measured in well D06 before the H 2 injection, but the mean values decreased over time, from 5.8 ± 0.0 (T0) to 3.4 ± 1.9 (injection) to 1.9 ± 0.4 (Post_injection).The lowest value of 1.4 was measured in well D06 5−6 days post injection.In the postincident phase, the values were higher again on average (4.3 ± 0.3).The data indicate that H 2 selectively favored the growth of certain prokaryotes capable of consuming H 2 , which then led to a decrease in α-diversity in well D06 (and to a lesser extent in D04) post injection.Supporting this hypothesis, the values were rather stable in the control well D11, ranging between 4.2 and 6.0 (5.4 ± 0.5) (Figure 1), indicating a moderate natural variability in α-diversity as previously observed at the test site. 26he NMDS (stress = 0.055) revealed that the samples taken from wells D04, D06, and D11 were distinct, confirming previous results, which showed that the natural groundwater microbiome on the test site is spatially and temporarily heterogeneous. 26Notably, the dissimilarities of the microbial communities from the individual wells temporarily increased due to the presence of H 2 , particularly in well D06 (Figure 2).Thus, the presence of H 2 caused a reduced α-diversity and a changing β-diversity in wells D04 and D06, indicating an enhanced growth of previously low-abundant community members in response to H 2 .
Dominant Taxa in Wells D04 and D06 Due to H 2 Injection, and Putative H 2 Oxidizers in Well D04.In well D04, the abundance of phylotypes affiliated to the genus Pseudomonas increased during H 2 injection and early postinjection, making up 63% of the microbial community at most (Figure 3A).Pseudomonas species are known to perform various metabolic processes, including H 2 oxidation. 10,11It might be that members of this genus oxidized H 2 in the initial phase after H 2 injection, meaning that O 2 was microbiologically consumed in addition to pure physical stripping by H 2 injection.However, we cannot rule out any community changes upon the injection phase resulting from displacement of water by the injection process itself; hence, water dominated by Pseudomonas phylotypes was transported to well D04.Later, i.e., 13−14, 19−20, 26−27, and 76−77 days after H 2 injection, Acetobacterium and Desulfovibrio were the most abundant genera in the groundwater microbial community of well D04 (Figure 3A).Members of both genera are known for the chemoautotrophic or chemoheterotrophic use of H 2 as an electron donor.Members of the genus Acetobacterium are obligately anaerobic and can oxidize H 2 while reducing CO 2 via formate to acetate. 40Desulfovibrio species are typical H 2 oxidizing sulfate reducers, and most strains require, when growing with H 2 as the energy source, acetate in addition to CO 2 as a carbon source. 41The presence of Acetobacterium and Desulfovibrio strongly suggests that H 2 was oxidized anaerobically using carbonate and SO 4 2− as electron acceptors until the beginning of the postincident phase; hydrogenotrophic acetogenesis was also indicated by transiently increasing the concentrations of acetate and formate (Figure S4A,E).Supporting this hypothesis, in microcosm experiments using sediment and groundwater from the test site which were supplied with H 2 , it was shown that homoacetogenesis via H 2 oxidation and CO 2 reduction was the dominant H 2 -oxidizing process at elevated H 2 levels. 18The acetate produced by Acetobacterium may have promoted the growth of Desulfovibrio, as previously observed in H 2 -driven sulfate-reducing bioreactors. 42,43Other sulfate-reducing phylotypes popping up post injection belonged to Desulfosporosinus and Desulfocapsaceae (Figure 3A), also indicating H 2 -driven growth.Notably, the increasing abundances of sulfate-reducing taxa in well D04 in the postinjection phase were not reflected by corresponding decreasing SO 4 2− concentrations; those remained at around 1.3 mM (11.5 m), 1.6 mM (14.5 m), and 1.9 mM (17.5 m) Figure 3. Heatmaps of the most common genera in the H 2 -exposed wells D04 (A) and D06 (B) as well as in the control well D11 (C).Taxa that occurred with >5% relative abundance in at least one sample of the total data set at the genus level were plotted for samples taken before (T0), during (Injection), and after (Post_injection) the H 2 injection, as well as when the H 2 concentrations in the H 2 -exposed wells fell below the detection limit (Post_incident).

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until 246 days after H 2 injection (Figure S3C), indicating that SO 4 2− concentrations were stratified before H 2 injection and remained stratified during and after H 2 injection and that SO 4 2− consumption and SO 4 2− supply were in the steady state in this well.Accordingly, sulfide, the product of dissimilatory sulfate reduction, was detected only sporadically and in low amounts (Figure S3D) and might have precipitated to metal sulfides in the sediment quickly after production; this assumption is supported by decreased concentrations of dissolved iron and manganese species at depths of 14.5 and 17.5 m in the postinjection phase (Figure S6A,B).In the last two groundwater samples collected from well D04 in the postincident period, Acetobacterium, Desulfovibrio, and Desulfocapsaceae phylotypes were less abundant, and the microbial communities became more diverse again (Figure 3A), indicating that specific H 2 consumers were slowly fading out.
Well D06.In well D06, the abundances of Pseudomonas phylotypes were slightly increased during the second day of the injection (see the discussion of well D04 above for possible reasons).On the third day of the injection as well as in the postinjection phase, the abundances of phylotypes affiliated with the genera Sulf uricurvum, Sulf urimonas, and Rhodoferax were strikingly increased (Figure 3B).Being facultatively anaerobic and chemolithoautotrophic, members of the genus Sulfuricurvum are known for the oxidation of sulfide, sulfite, elemental sulfur (S 0 ), thiosulfate, and H 2 with O 2 (microaerobic conditions) as well as NO 3 − as electron acceptors. 44,45ulfurimonas species are widespread and aerotolerant to being facultatively anaerobic.Besides growth on organic compounds, they grow chemolithotrophically with reduced inorganic sulfur compounds or H 2 as electron donors, NO 3 − , NO 2 − , or O 2 as electron acceptors, and CO 2 or organic compounds like acetate as carbon sources. 46,47Hence, the notably high abundances of phylotypes affiliated to Pseudomonas and especially to the genera Sulfuricurvum and Sulfurimonas suggest that H 2 was oxidized by members of these genera in well D06 during the injection as well as in the postinjection phase, using NO 3 − and O 2 as electron acceptors.Increasing abundances of Acetobacterium in the late postinjection phase indicated anoxic conditions and H 2 consumption by homoacetogens, similar to well D04, and supported by the detection of acetate and formate (Figure S4E,F).In contrast to well D04, typical sulfate reducers were not abundant in the postinjection phase, indicating that sulfate reduction was not an important electron-accepting process for H 2 oxidation around well D06.The increased abundances of Rhodoferax phylotypes could have been promoted by increased acetate concentrations 48 provided by homoacetogens; on the other hand, members of the Rhodoferax genus may have also oxidized H 2 using Fe 3+ as an electron acceptor. 49Post incident, Sulf urimonas and especially Sulf uricurvum became less abundant and the diversity of the microbial communities increased again, with Desulfosporosinus and Candidatus_Accumulibacter being the most abundant taxa (Figure 3B).Composition of Communities Not Affected by the Presence of H 2 .At the class level, the unaffected microbial communities (samples taken from wells D04 and D06 before H 2 injection and all samples from the control well D11) were predominantly composed of Gammaproteobacteria, Bacteroidia, Parcubacteria, and Alphaproteobacteria (Figure S8) as previously observed at this site. 26−53 At the family or genus level, prior to the injection, the microbial communities in all wells were composed of various taxa, with none of them occurring with an abundance >15%.Phylotypes related to the family MA-28-I98C (belonging to the order Desulfuromonadales) and the genera Pseudomonas and Gallionella were the most abundant taxa.
In control well D11, the compositions of the microbial community were rather stable during the whole investigated time frame, except that phylotypes belonging to Pseudomonas, Massilia, and Oxalobacteraceae were highly abundant in the samples taken on the third injection day as well as 5−6 days post injection.Notably, these genera also showed increased

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abundances in well D06 on the second day of the injection, as well as in well D04 on the third day of the injection.While microbial community members potentially capable of H 2 oxidation quickly became dominant in the H 2 -exposed wells (see the discussion above), the abundances of Pseudomonas, Massilia, and Oxalobacteraceae seem to continually increase in well D11 from the second day of the injection to 5−6 days after the H 2 injection.The sample collected 5−6 days post injection additionally showed the lowest value in α-diversity and the highest dispersion in β-diversity in well D11 (Figures 1  and 2).This indicated that the gas injection with the concomitant increase in the pressure head leads to a displacement of the surrounding waterbody, hypothesized already by the geochemical data. 18Nevertheless, the H 2 concentrations were below the detection limit in well D11 during the entire monitoring period.−58 Total Cell Count Changes Due to H 2 Injection.The total cell counts in samples taken before the H 2 injection were between 1.3 × 10 5 and 3.8 × 10 5 cells/mL, which is within the range determined at natural conditions (3.2 × 10 4 −2.0 × 10 6 cells/mL) in our previous study 26 and, furthermore, within the range that would be expected for pristine groundwater, i.e., 10 4 −10 6 cells/mL. 59During and after the H 2 injection, the values determined in samples from the H 2 -exposed well D04 and the control well D11 did not rise above 4 × 10 5 cells/mL, but those from the H 2 -exposed well D06 increased up to (1.2 ± 0.3) × 10 6 cell/mL 5−6 days post injection (Figure 4).This number is still within the normal range of the 10 4 −10 6 cells/ mL expected in pristine groundwater.The elevated total cell counts in well D06 detected after H 2 injection could be due to the stimulated growth of Sulfuricurvum and Sulf urimonas.H 2 oxidation using O 2 or NO 3 − as the electron acceptor is energetically more favorable than sulfate or carbonate-dependent H 2 oxidation, 8 which could explain why significantly increased cell counts were measured in well D06 but not in well D04, where microbial community data suggest predominantly anaerobic H 2 oxidation by Acetobacterium and Desulfovibrio.
Environmental Implication of Gaseous H 2 Injection.In summary, the data demonstrate that the injected H 2 was rapidly oxidized by various indigenous, ecophysiologically different microorganisms, leading to decreased α-diversity and concurrently changed β-diversity.In well D04, increased abundances of sulfate-reducing and acetogenic taxa indicated H 2 consumption primarily by the use of SO 4 2− and CO 2 as electron acceptors, respectively.In well D06, high abundances of aerobic, nitrate-reducing, iron-reducing, and (later) acetogenic taxa indicated H 2 oxidation primarily by the use of O 2 , NO 3 − , Fe 3+ , and later CO 2 as electron acceptors.The presented data confirm the results of previously published δ 2 H analyses of H 2 , which indicated microbial consumption of H 2 in wells D04 and D06. 18The assumed consumption of H 2 by microbes using different electron acceptors (O 2 , NO 3 − , Fe 3+ , SO 4 2− , or CO 2 ) indicated by the microbial community analyses was not concomitant with a corresponding clear in situ depletion of all of these electron acceptors in the investigated wells, although the aquifer was locally saturated with H 2 during injection.Hence, oxidation of H 2 , reduction of electron acceptors, and subsequent supply of electron acceptors by groundwater flow may have been in a steady state; furthermore, end products of electron acceptor reduction were likely transformed (acetate) or precipitated (FeS).Considerable amounts of H 2 might have been distributed in the aquifer by physicochemical processes, perhaps resulting in outgassing of an unknown amount of the injected H 2 .Notably, the H 2 -induced effects on the microbial community were shown to be temporarily limited as αand β-diversity approached the initial state again at the end of the monitoring period, resulting in no persistent negative effects on the groundwater microbiome in the course of the investigated underground H 2 leakage scenario.On the other hand, in the case of a continuous H 2 leak from an underground storage site, spatially separated microbial communities likely develop alongside the H 2 plume depending on the availability of electron acceptors, a scenario that is well-known in aquifers contaminated by aromatic hydrocarbons degradable with various electron acceptors. 60ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.3c04340.

Figure 1 .
Figure 1.Shannon−Wiener indices showing the α-diversity of the microbial communities in wells D04, D06, and D11 sampled before (T0), during (injection), and after (Post_injection) the H 2 injection.The H 2 concentrations in the H 2 -exposed wells fell below the detection limit in the postincident phase (Post_incident).

Figure 2 .
Figure 2. β-diversity illustrated by nonmetric multidimensional scaling (NMDS) using a Bray−Curtis dissimilarity matrix.The microbial communities in the H 2 -exposed wells D04 and D06 and the control well D11 were distinct and, furthermore, microbiome dispersion temporarily changed, especially in well D06 during the injection and postinjection phase.

Figure 4 .
Figure 4. Total cell counts of samples from the H 2 -exposed wells D04 and D06 as well as the control well D11 taken before (T0), during (Injection), and after (Post_injection) the H 2 injection, as well as when the H 2 concentrations in the H 2 -exposed wells fell below the detection limit (Post_incident).The H 2 concentrations are also shown (black dots).